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Vol. 7 No. 1 (2015)

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University . Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net

Citation :Egypt.Acad.J.Biolog.Sci. ( C.physiology and Molecular biology ) Vol.7(1)pp39-49 (2015) . Acad. J. Biolog. Sci., 7(1): 39 - 49 (2015) Egyptian Academic Journal of Biological Sciences C. Physiology & Molecular Biology ISSN 2090-0767 www.eajbs.eg.net

Molecular phylogenetic relationship among three (Reptilia: ) in Egypt based on 18S rRNA Sequences

Moustafa Sarhan1, Islam Saber1, Mohamed El-Shehaby1 and Samy Saber2 1- Department of Zoology, Faculty of Science, Al-Azhar University, Assiut. 2- Department of Zoology, Faculty of Science, Al-Azhar University, Cairo.

ARTICLE INFO ABSTRACT Article History Acanthodactylus is a genus of lacertid , commonly Received: 27/3/2015 referred to as fringe-fingered or fringe-toed lizards. The Accepted:4/5/2015 systematics of the genus Acanthodactylus was classically based on ______external morphological traits, osteological characters and Keywords: morphology of the hemipenes. In this study, partial sequences of Lacertidae, Acanthodactylus 18S rRNA gene were used to estimate the relationships among 18S rRNA three lacertide species from Egypt: Acanthodactylus boskianus, Phylogeny Acanthodactylus scutellatus and Acanthodactylus longipes. These Sequencing data were used together with morphological information, to evaluate the relationships among these three species. DNA was extracted from the collected specimens, 988 bp fragment of 18S rRNA was amplified using universal primers and sequenced. The obtained sequences were analyzed and a phylogenetic tree was constructed. Results of analysis revealed that A. scutellatus and A. longipes form a very well supported clade with 86% bootstrap. A. boskianus was found to constitute a separate clade. The resultant tree based on DNA sequence data was similar to that produced from a differentially weighted data set of morphological characters. In conclusion, the 18S rRNA gene sequence seems to be a good marker for phylogenetic analysis of Acanthodactylus species.

INTRODUCTION Lizards of the family Lacertidae are widely distributed throughout most of Eurasia and all of . Presently, approximately 259 species of lacertid lizards have been assigned to 24 genera (Fu, 1998). The genus Acanthodactylus Fitzinger, 1834, is commonly known as the fringe-fingered lizards and is the largest genus in the family Lacertidae with over 40 described species (Uetz, 2013) and 36 species listed in the EMBL Database (http://www.embl-heidelberg.de/~uetz/ LivingReptiles.html) Lizards of this genus constitute an important part of the vertebrate fauna in many arid ecosystems in the Middle East and North Africa (Nouira et al., 1994, Shenbrot et al., 1997), where they are often the most conspicuous diurnal . As such, they have been the subjects of many ecological (Aljohany et al., 1989, Mellado et al., 1991, Mellado et al., 1992, Perry et al., 1994, Belliure et al., 1996) and behavioural (Perry et al., 1990, Day et al., 1999, Sword et al., 2000) studies.

Citation :Egypt.Acad.J.Biolog.Sci. ( C.physiology and Molecular biology ) Vol.7(1)pp39-49 (2015) 40 Moustafa Sarhan et al.

Most of the Acanthodactylus Schleich et al., 1996, Rastegar et al., species inahbit the southern side of the 1999, Sindaco et al., 2000, Sindaco et al., Mediterranean basin and the Arabian 2008). The only molecular phylogenetic Peninsula. One species reaches Europe study on the entire Acanthodactylus (Iberian Peninsula), and the genus range genus, however, was published by Harris extends east to western India and south to & Arnold (2000), who suggested that the the Sahel zone in Africa (Salvador, A. scutellatus group includes A. 1982). Four fundamental studies scutellatus and A. longipes, while A. constructed available systematic boskianus constitute a separate group. knowledge of Acanthodactylus, based on The A. scutellatus group is a external morphology, osteological monophyletic clade consisting of several characters, and the morphology of the sand adapted species found in North hemipenes (Boulenger, 1918, Salvador, Africa and northern Arabia. 1982, Arnold,1983, and Harris et al, Acanthodactylus longipes was described 2000). The latter three studies divided the as a variety of A. scutellatus. It was first genus into species groups, a division that recognized as a full species by Bons & is commonly used today, although the Girot (1962), based on its sympatric assignment of some species to groups is occurrence with other members of the A. debated (Acanthodactylus blanfordii scutellatus group in various parts of its Boulenger, 1918, and Acanthodactylus range. The specific status of A. longipes masirae Arnold, 1980, Harris et al., is now widely acknowledged (Salvador 2000). The systematics of some species 1982, Arnold 1983, Crochet et al., 2003). groups is unclear and unstable because of Baha el din (1994, 1996) was first high intraspecific variability of some reported the species from Egypt, which species and morphological convergence had been previously overlooked because among species (the description of of its close similarity to A. scutellatus. Acanthodactylus mechriguensis Nouira et Subsequently other authors adopted this al., 1999, Fonseca et al., 2008). The designation for relevant populations in distinction among the species within Egypt and Israel (Saleh 1997, Werner Acanthodactylus using superficial 1998, Bouskila & Amitai 2001). The features is taxonomically difficult. This is previous molecular studies on due to the considerable variation in the Acanthodactylus systematic were based range of external characters such as body on mitochondrial genes (e.g. 12S, 16S size, size of dorsal scales and absence of and cytochrome b) and nuclear genes as enlarged dorsal tubercles when present, in Tamar et al.2014. In this study, we their number, size and shape and other used 18S rRNA for the phylogeny of morphological characters (Tamar et al, Acanthodactylus species. In fact, 2014).Thus, the most problematic and Ribosomal RNA is considered as the best interesting issues in Acanthodactylus target for studying phylogenetic systematics are the relations amongst and relationship because, it is universal and is within species groups, the of composed of highly conserved as well as the genus, and its biogeography. variable domains (Woese et al., 1987, Acanthodactylus boskianus is the Goerge et al., 1977). The ribosomes most widespread species of its genus (׽8 consist of rRNA and proteins. In all 000 000 km2; S. Meiri, unpublished organisms the ribosome consists of two data), ranging throughout North Africa subunits, the small ribosomal subunit and the Sahel, the whole Arabian (SSU) contains a single RNA species (the Peninsula, eastwards to Iran, and 18S rRNA in eukaryotes and the 16S northwards to Turkey (Salvador, 1982, rRNA in others). Moreover, rRNA genes Molecular phylogenetic relationship among three Acanthodactylus species in Egypt 43 are evolving more slowly than protein following manufacturer's instruction. encoding genes and are particularly DNA was then eluted in 200 µl Tris- important for the phylogenetic analysis EDTA (TE) buffer. The quality of the of distantly related species (Moritz et al. genomic DNA was examined on 0.8% 1987). agarose gels stained with ethidium In order to clarify the systematics bromide (10µg/ml) (Sambrook et al. and to reveal the phylogenetic 1989) and OD260 was determined by relationships between A. boskianus, A. Nanodrop. scutellatus and A. longipes in Egypt, Amplification of target fragment: fragments of ribosomal gene 18S rRNA PCR primers pair designated 18S (18S) is herein sequenced and analyzed rRNA F 5’- for genetic variation. This study TTAAGCCATGCATGTCTAAG-3’ introduced the 18S rRNA gene sequence and 18S rRNA R 5’- as a good marker for phylogenetic GACTACGACGGTATCTAATC-3’, analysis of Acanthodactylus species in synthesized by Metabion International addition to the morphological characters. AG (Martinsried/Deutschland) were used. Polymerase chain reaction (PCR) MATERIALS AND METHODS with universal primers situated in the Specimens: flanking regions of the target fragments A total of 40 specimens belonging was performed to amplify the fragments to the three Acanthodactylus species; A. of interest. PCR was performed in a 25 longipes, A. boskianus, and A. scutellatus µl volume of solution containing 12.5 were collected from Marsa matruh, µl Go Taq Green Master mix (promega), Baltim and North Sinai respectively were 2 µl of primer F, 2 µl of primer R used in this study (Fig.1). Species as well (10pmoles), 3.0 µl of template DNA(100 as collection date and locality for each ng) and completed with nuclease free specimen were recorded. In the water to reach the required volume. laboratory, sex of each of the collected The PCR reaction was performed in a specimens of each species was mini thermocycler (Techgene) using the determined on the basis of morphologic following cycling protocol: initial criteria given by Schleich et al. (1996). denaturation at 94oC for 2 min. and then In some cases sex was ascertained by 35 cycles of 94oC for 30 sec, 52oC for 30 opening the abdomen and checking the sec, and 72oC for 3 min. Final extension gonads. was carried out at 72oC for 10 min. Morphological analysis: Amplified products from the PCR were Morphological traits examined electrophoresed on 1.5% agarose gels consist of mensural and meristic stained with ethidium bromide. A 100 bp characters and are summarized in Fig.2. ladder (Jena Bioscince, GmbH, Mensural measurements were taken Germany) was loaded in each gel then conventionally using a mm ruler and photographed under UV light with a gel calipers. Spss 17.0v software was used to documentation system. assess and compare the taxonomic Sequencing of 18S rRNA Gene distances between the three species. Products: DNA Extraction: Each of PCR-product was purified Genomic DNA from specimens of with QIAquick-spin PCR purification kit the three Egyptian Acanthodactylus (Qiagen,Germany). A second PCR was species was extracted using a DNeasy then performed using BigDye Terminator blood and tissue kit (QIAGEN) for v3.1 cycle sequencing Kit, for a total purification of total DNA from volume of 20 µl, with each reaction tissues (Spin-Column Protocol) contained 8 µl Terminator ready reaction 44 Moustafa Sarhan et al. mix, 3.2 pmol primer, DNA template AY217917) was included by Basic Local (template quantity was calculated Alignment Search Tool (nBLAST) according to the PCR product size) and (www.ncbi.nih.gov/BLAST/) in the deionized water. Thermal profile for NCBI database (National Center for Cycle Sequencing PCR was 1 min at Biotechnology Information, NIH, 96oC, 25 cycles (10 sec. at 96oC, 5sec. at Bethesda, Maryland, USA) (Tatusova 50oC, 4 min at 60oC). After additional and Madden 1999). Bootstrap analysis step of purification with CENTRI-SEP employed 500 replicates. For the 18S Columns (Prenciton Separation), DNA rRNA gene, the JC (Jukes-Cantor) + G sequencing was applied by 3500 genetic (gamma) model of nucleotide analyzer, Applied Biosystems. At least substitution was determined as the most two independent PCR products were appropriate model according to Bayesian used for sequencing per species. Information Criterion (BIC) and Akaike Sequences Analysis: Information Criterion (AIC) (Nei and Chromatograms and sequences Kumar 2000). The analysis involved 4 were analyzed and edited using BioEdit nucleotide sequences. Codon positions program®, version 7.2.5 (Hall 1999). included were 1st+2nd+3rd+Noncoding. The obtained DNA sequences were All positions containing gaps and aligned using Basic Local Alignment missing data were eliminated. There were Search Tool software [BLAST, version a total of 954 positions in the final 2.2.22 (http://blast.ncbi.nlm.nih.gov). All dataset. Genetic distances were created in new DNA sequences reported in this MEGA6 software paper were deposited in GenBank (http://www.megasoftware.net). sequence database provided by the National Center for Biotechnology RESULTS Information (accession numbers; Morphological analysis: KR054729, KR054730, KR054731, Differences and similarities among KR054732, KR054733). Multiple these species are assessed using alignment analysis was performed with phenogram analysis based on the CLUSTALW computer program morphological taxonomic characters. (Thompson et al 1994) in MEGA6 Phenogram (Fig. 3) shows differences software (http://www.megasoftware.net). and similarities between three species of Phylogenetic construction: Acanthodactylus. The phenogramp UPGMA and Maximum Parsimony clearly shows the morphological trees were constructed using MEGA6 similarity, as indicated by the short software (http://www.megasoftware.net). taxonomic distance between the two In order to improve the homology closely related species A. scutellatus and statements, outgroup (Mesalina A. longipes, and a longer distance guttulata) (Accession number between these and A. boskianus. PCR Products of 18S rRNA gene and The data set of this study is comprised of sequence data of the Acanthodactylus 4 sequences of A. boskianus, A. species: scutellatus, A. longipes and the outgroup PCR products were amplified from (Mesalina sp.). The data set included ribosomal gene using synthesized ribosomal gene fragments of 18S (׽961 primers set. Across the alignment of 18S bp). The number of variable (V) sites rRNA, long region was suitably were 30, parsimony-informative (Pi) sites conserved to allow primer design. The were 8, conserved sites were 928 (Fig. 5). PCR products were approximately 1000 Nucleotide composition were T (22.7%), bp for 18S rRNA gene of A. boskianus, C (25.3%), A (24.0%) and G (28.1%). A. scutellatus, and A. longipes (Fig. 4). The Overall average of genetic distance Moustafa Sarhan et al. 40 was 0.019. The level of genetic distance comprises Acanthodactylus sp., clade (B) (p-distance) in the 18S rRNA between constitutes outgroup (Mesalina sp.). In species is shown in Table 1. The table clade (A), A. scutellatus and A. longipes shows a genetic distance of 1.4% form a very well supported clade (high between A. scutellatus and A. longipes, value of bootstrap (86%). While A. 2.0% between A. boskianus and A. boskianus constitutes a separate clade. longipes and and 2.3% between The three Egyptian Acanthodactylus Acanthodactylus sp. and the outgroup. gathered in one cluster forming one clade Phylogenetic construction: separated from the lacertidae outgroup. The above analysis resulted in a Egyptian A. boskianus is clearly topology with a mean log-likelihood genetically distinct from other two values lnL= -1610.894, BIC = 3271.294, species. Furthermore, UPGMA method AICc = 3233.809, nucleotide resulted in the same phylogeny as in frequencies: f (A)= 0.250, f (C)= 0.250, f Maximum Parsimony method, with A. (G)= 0.250, f (T)= 0.250. Our scutellatus and A. longipes being more phylogenetic analysis using Maximum genetically related to each other than to Parsimony method distinguishes two A. boskianus. major clades (Fig. 6): clade (A)

Table 1: Average uncorrected genetic distances (p-distance) between species clades of Acanthodactylus from Egypt based on 988 bp fragment of 18S rRNA. A .boskianus A .longipes 0.020 A. scatellatus 0.015 0.014 Mesalina guttulata (outgroup) 0.018 0.023 0.023

DISCUSSION In conventional, morphology-based Classical taxonomy relies primarily Acanthodactylus systematics is fairly on morphological characteristics to easy to assign species to species groups. elucidate the phylogenetic relations However, the boundaries between among organisms. Recently, the species and relationships within species molecular approach based on comparing groups are often unclear and unresolved nucleotide sequences of RNA and DNA (Salvador, 1982, Arnold, 1983, Harris et and sequences of amino acids of protein al., 2000, Crochet et al., 2003, Harris et open a new era in phylogenetic analysis. al., 2004, Fonseca et al., 2008, 2009). Both the classical morphology-based These variations make it hard to methods and molecular taxonomy construct clear species identification methods are of importance as the basic keys. In Acanthodactylus, as in many bio-molecular framework of all animal groups, molecular phylogenetic organisms are similar and morphology of analysis offers a decisive method to an organism is actually the distinguish species and their taxonomic manifestations of its genome, proteome relations. Our results, based on 18S and transcriptome profiles. A rRNA data reveal that A. scutellatus and combination of the morphological based A. longipes are monophyletic and A. methods and molecular analysis based boskianus was genetically distinct from methods, thus strengthens the exercise of other species used in this study, with A. the determination of phylogenetic boskianus forming a separate clade. relationships of organisms to a great The 18S rRNA molecule of the extent (Patwardhan et al. 2014) ribosomal small subunit is frequently used to infer phylogenetic affiliations of 45 Molecular phylogenetic relationship among three Acanthodactylus species in Egypt ancient (>100 Mya) eukaryotic relationships. Its suitability as a REFERENCES phylogenetic tool is two-fold. First, it is a Aljohany A.M.H., Spellerberg I.F. good source of phylogenetic information (1989). Home range and vagility of based on conservation of function, the lizards Acanthodactylus variable mutation rates depending on schmidti and Acanthodactylus substructure position, and its ubiquity in boskianus. Saudi Arabia. Journal all taxa. Second, the molecule provides of Arid Environments 16: 79–86. readily obtainable nucleotide sequences Arnold E.N. (1980). The scientific results because of high rRNA transcript copy of the Oman flora and fauna survey number in eukaryotes and ease of PCR 1977 (Dhofar). The reptiles and primer design (Woese, 1987Kjer, 1995). amphibians of Dhofar, southern The 18S rRNA molecule contains certain Arabia. Journal of Oman Studies stable sections having low substitution Special Report 2: 273–332. rates. As such, 18S rRNAs can provide Arnold E.N. (1983). Osteology, genitalia informative characters for assessing and the relationships of affiliations of evolutionarily distant taxa. Acanthodactylus (Reptilia: However, 18S rRNAs also possess Lacertidae). Bulletin of the British variable regions having high substitution Museum (Natural History) Zoology rates. In some organisms these rapidly 44: 291–339 evolving expansion regions (or helices) Baha El Din S.M. (1994). A contribution provide phylogenetic signals for to the herpetology of Sinai. British discerning relationships between Herpetological Society Bulletin 48: evolutionarily closer clades. Calculated 18–27. genetic distance among the three studied Baha El Din S.M. (1996). On the species provide a molecular support to occurrence of Acanthodactylus conventional phylogenetic relationship longipes Boulenger, 1918, in among Acanthodactylus sp. Egypt, with remarks on its identification and ecology. Zoology CONCLUSION in the Middle East 12: 53–58. Molecular data provide Belliure J., Carrascal L.M. (1996). independent evidence that the differential Covariation of thermal biology and weighting of morphological characters in foraging mode in two past analyses was appropriate, where A. Mediterranean lacertid lizards. longipes and A. scutellatus are Ecology 77: 1163–1173. monophyletic group. The 18S rRNA Bons J. & Giort B. (1962). Revision of gene sequence is shown to be a good the species Acanthodactylus marker for supra-specific differentiation scutellatus (Lacertidé-Saurian). and for taxa grouping purposes. Natural Science Society and However, additional species and/or Physsiques of 42: 313- additional genes need to be studied for 334 assessment of the true placement of all Boulenger G.A. (1918). On the lizards of the Acanthodactylus species within the genus Acanthodactylus Wieg. Egypt. Bulletin of the Zoological Society of France 43: 143-155. ACKNOWLEDGMENTS Bouskila A. & Amitai P. (2001).A The authors are grateful to Prof. handbook of the reptiles and Mostafa Saleh (Zoology Department, amphibians of Israel [in Hebrew]. Faculty of Science, Al Azhar University) Jerusalem, 345 pp. for reviewing the manuscript. Moustafa Sarhan et al. 46

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Fig. 1: General view of three Acanthodactylus sp. from Egypt.

Fig. 2: Morphological characteristics used for the identification of Acanthodactylus species. A. Body measurements; Example of the snout to vent length (SVL) measurement. B. Head measurements.

Fig. 3: A phenogram showing the average taxonomic distance (dissimilarity) between three species of Acanthodactylus from Egypt.

Citation :Egypt.Acad.J.Biolog.Sci. ( C.physiology and Molecular biology ) Vol.7(1)pp39-49 (2015) 48 Moustafa Sarhan et al.

Fig. 4: PCR products (~1000bp fragment) of 18s rRNA gene amplified by universal primers pair from Egyptian lacertides of Acanthodactylus boskianus, Acanthodactylus scutellatus, Acanthodactylus longipes and 100 bp DNA Ladder Marker.

Fig. 5: Nucleotides multiple alignment of partial 18S rRNA gene sequences of three Egyptian Acanthodactylus. Colored columns represent identical nucleotide sequences between aligned isolates.

Molecular phylogenetic relationship among three Acanthodactylus species in Egypt 49

Fig. 6: Rooted phylogenetic tree among the three Egyptian Acanthodactylus sp. based on ribosomal gene (18S rRNA) using Maximum Parsimony method.